DESCRIPTION: The broad objective of this proposal is to understand the molecular mechanisms cells use for removing DNA damage. The selective removal of DNA damage from the transcribed strands of active genes, termed transcription-coupled nucleotide excision repair (TCNR), may be ubiquitous and has been clearly demonstrated for the removal of ultraviolet light induced cyclobutane pyrimidine dimers. This research is focused on characterizing the mechanism, generality and biological consequences of transcription-coupled repair. Studies in E. Coli have been featured because of the availability of mutants that we have used to identify genes required for the process. The researchers will continue to study TCNR in E. Coli and will complement these studies with the examination of TCNR in human cells. Approaches to be taken include the following: (1) Determine how components of mismatch repair influence nucleotide excision repair inE. Coli using genetic and biochemical approaches. To study the potential biochemical interactions between components of mismatch repair and components of nucleotide excision repair, we will attempt to establish a system in which we can study TCNR in E. Coli in vitro. (2) Establish that mutations in human mismatch repair genes influence the removal of cyclobutane pyrimidine dimers from the DHFR gene in tumor cell lines and lymphoblastoid cell lines from patients with hereditary nonpolyposis colorectal carcinoma (HNPCC). If mutations in human mismatch repair genes also abolish transcription-coupled repair, then the etiology of tumors associated with mutations in mismatch repair genes may be influenced by deficiencies in nucleotide excision repair and base excision repair, in addition to the deficiency in mismatch correction. (3) The researchers will also attempt to determine the mechanism by which mutations in the gene encoding the beta subunit of the E. Coli RNA polymerase dramatically reduce nucleotide excision repair in nontranscribed DNA. These mutants pheontypically resemble human xeroderma pigmentosum group C cell lines.